Four different phosphatase activities were isolated from turkey gizzard smooth muscle. Phosphatase I is a trimer which dephosphorylates myosin light chain kinase, resulting in activation of this enzyme. The catalytic subunit of phosphatase I, but not the holoenzyme, can dephosphorylate myosin, resulting in inhibition of the actin-activated MgATPase activity. phosphatase II is purified from other sources, such as cardiac muscle. Phosphatase III, which can be separated from phosphatase I by hydrophobic chromatography, is active in dephosphorylating myosin and appears to be substrate specific. Phosphatase IV, which dephosphorylates both myosin and the isolated light chain of myosin may be a product of phosphatase III.